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Dissertation / PhD Thesis/Book | PreJuSER-1305 |
2008
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/13949
Report No.: Juel-4276
Abstract: During the past few years detailed knowledge of the structure of sensory rhodopsin II (NpSRII) and its cognate transducer HtrII from Natronobacterium pharaonis has been gained. NpSRII, a member of the rhodopsin family, serves as a model protein for G-protein coupled receptors. To increase the state of knowledge of the relations between structure and function, earlier studies determined the strength of NpSRII/NpHtrII-binding in detergent buffer. However, little is known about the complex formation in more physiological conditions like in lipid membranes. Binding studies for low protein concentrations as they occur in N. pharaonis have not been performed to this date. The evaluation of NpSRII/NpHtrII-binding was implemented by measuring Förster resonance energy transfer (FRET) of fluorescently labelled proteins at different concentrations. In this work the dissociation constant of NpSRII/NpHtrII in detergent buffer (KD = 200 nM) was reproduced and the complex binding in large unilamellar vesicles (LUV) was analyzed, subsequently. A comparison of FRET-efficiencies, as observed for proteins in detergent buffer and in lipid membranes, shows increased binding affinities of NpSRII and NpHtrII in lipids of at least one order of magnitude. Hence, these results support previous studies, which show that the results observed in detergent buffers are limited in their informational value. In further studies the proteoliposomes were incorporated into giant unilamellar vesicles (GUV) by peptide-induced membrane fusion, to mimic physiologically low protein concentrations. This led to a molar protein-lipid-ratio of 1:1,000,000, as it occurs in bacteria. From this model system exact diffusion coefficients of fluorescently labelled NpSRII and NpHtrII were determined by using the new 2fFCS-technique (two-focus fluorescence correlation spectroscopy). The results indicate, that the NpSRII/NpHtrIIbinding is observed even at physiologically protein concentration. The diffusion coefficients were proportional to the inverse cylinder radius of the proteins. This is in the line with recently published studies that show discrepancies to the far spread model of Saffman and Delbrück.
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